Cultures of dopaminergic neurons from the mesencephalon of embryonic rat were grown in fully defined Neurobasal/B27 supplemented serum-free medium. When exposed to increased extracellular K+ concentrations, these cultures are damaged mainly via NMDA-receptors. Because in vivo cells are exposed to high concentrations of extracellular K+ during ischemia, anoxia, or seizures, we investigated the mechanisms involved in K+-induced neurotoxicity and compared them to those involved in NMDA-induced neurotoxicity. Damage following exposure to high K+ levels is blocked by MK-801 (2.5uM), indicating a role for NMDA receptors. CNQX does not protect neurons from high K+-induced damage. This damage does not occur in Ca2+-free medium, and the Ca2+/calmodulin antagonist W-5 at 150-200 uM completely protects neurons from the damage. If KN-62, an inhibitor of calmodulin-dependent Protein Kinase II is present at 1 muM, about 60-70% of neurons survive, but if, in addition, cyclosporin A, an inhibitor of the pritein phosphatase, calcineurin, is present in medium at 1 muM, only 30-40% of the neurons survive. Because these findings implicated Ca2+ and Ca2+/calmodulin dependent mechanisms in the high K+-induced neurotoxicity, we examined further their possible relationship glutamate-induced toxicity. W-5 (50 and 100muM) reduced K+-induced glutamate release by 30-50%. At 200 uM W-5 delayed glutamate release, but glutamate levels eventually reached those seen in the absence of W5. KN-62 reduced glutamate release by 30-50% at 0.2 and 5 uM and 100% at 1 uM . The calcineurin inhibitor cyclosporin A alone only slightly glutamate release, but together with KN-62 delayed glutamate release and reduced its final level to 40% of that seen with KN-62 alone. It appears that the lutamate release is triggered by activation of calmodulin by Ca2+ entering the cells. The earliest response to calmodulin activation is calcineurin-catalyzed protein dephosphorylation. Eventually the extracellular glutamate concentration is regulated by the balance of the activities of the two calmodulin-dependent enzymes; Ca2+/ calmodulin- dependent protein kinase II and calcineurin.